The Na/Ca exchanger couples the movement of Ca ions across the plasma membrane to the movement of Na ions in the opposite direction. It is the principal Ca efflux mechanism in cardiac myocytes and plays an important role in controlling the Ca content of myocardial cells and in regulating cardiac contractility. Little is known about the cellular mechanisms that regulate exchange activity. In this project, the general hypothesis to be investigated is that sphingolipids are physiological regulators of Na/Ca exchange activity and block the conformational transition associated with regulatory Ca activation of the exchanger. This hypothesis is based on preliminary results demonstrating that short chain ceramide analogs and sphingosine inhibit exchange activity in a manner that is specifically targeted to the exchanger's regulatory properties. Exchange activity will be measured using fluorescent cytosolic Ca indicators in transfected CHO cells expressing the cardiac Na/Ca exchanger and in neonatal rat cardiac myocytes. The project has 6 specific aims: (1) Initial characterizations of the effects of exogenous and endogenous ceramide or sphingosine on exchange activity in the transfected cells will be completed. (2) A new kinetic formulation of exchanger regulation, which specifically links the process termed "Na-dependent inactivation" to changes in regulatory Ca-activation, will be developed. (3) The rate at which ceramide inhibition develops when cytosolic Ca is elevated or when internal Ca stores are filled will be measued in order to test the hypothesis that ceramide inhibition requires the formation of the Ca-depleted conformation of the exchanger. Other studies will test the prediction that ceramide does not inhibit the exchanger in the absence of Ca activation. (4) The hypothesis that ceramide inhibits exchange activity by perturbing lateral lipid subdomains such as lipid rafts and/or caveolae will be tested. (5) The studies in this aim will test the idea that pharmacological stabilization of the actin cytoskeleton locks the exchanger in an activated state, reducing its susceptibility to regulation by cytosolic Ca or inhibition by ceramide. (6) The physiological significance of ceramide inhibition of exchange activity will be assessed by measuring the influence of ceramide on (a) exchange activity, (b) the Ca-transient and (c) the amount of Ca in the sarcoplasmic reticulum in beating neonatal rat cardiac myocytes. Other studies will assess whether inhibition of exchange activity contributes to the cardiotoxic effects of TNFalpha.